U.S. patent application number 11/286237 was filed with the patent office on 2006-04-13 for methods of treating and preventing bone loss.
Invention is credited to John David Allard, Robert Frederick Klein, Gary Allen Peltz.
Application Number | 20060079488 11/286237 |
Document ID | / |
Family ID | 27734489 |
Filed Date | 2006-04-13 |
United States Patent
Application |
20060079488 |
Kind Code |
A1 |
Allard; John David ; et
al. |
April 13, 2006 |
Methods of treating and preventing bone loss
Abstract
Methods of treating and preventing bone loss and/or enhancing
bone formation are disclosed. The methods utilize 15-lipoxygenase
inhibitors. These molecules can be delivered alone or in
combination with agents which inhibit bone resorption or additional
agents that regulate calcium resorption from bone or enhances bone
accumulation. The invention additionally provides methods of
diagnosing a predisposition to bone loss.
Inventors: |
Allard; John David;
(Milpitas, CA) ; Klein; Robert Frederick;
(Portland, OR) ; Peltz; Gary Allen; (Redwood City,
CA) |
Correspondence
Address: |
ROCHE PALO ALTO LLC;PATENT LAW DEPT. M/S A2-250
3431 HILLVIEW AVENUE
PALO ALTO
CA
94304
US
|
Family ID: |
27734489 |
Appl. No.: |
11/286237 |
Filed: |
November 23, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10361093 |
Feb 7, 2003 |
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11286237 |
Nov 23, 2005 |
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60355255 |
Feb 8, 2002 |
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Current U.S.
Class: |
514/102 ;
435/6.16 |
Current CPC
Class: |
A61P 19/00 20180101;
A61K 38/23 20130101; G01N 2333/90241 20130101; A61K 2300/00
20130101; A61K 2300/00 20130101; A61K 2300/00 20130101; A61K
2300/00 20130101; A61K 2300/00 20130101; A61K 2300/00 20130101;
A61K 2300/00 20130101; A61K 2300/00 20130101; A61K 38/30 20130101;
A61K 31/192 20130101; A61K 31/56 20130101; A61K 38/30 20130101;
A61K 38/23 20130101; A61K 31/192 20130101; A61P 19/02 20180101;
A61K 31/404 20130101; C12Q 1/26 20130101; A61P 19/10 20180101; A61K
31/167 20130101; A61K 31/663 20130101; A61K 31/167 20130101; A61K
31/407 20130101; A61P 19/08 20180101; A61K 31/56 20130101; A61P
1/02 20180101; A61K 31/404 20130101; G01N 2800/108 20130101; A61K
31/663 20130101; G01N 2500/04 20130101; A61K 31/407 20130101 |
Class at
Publication: |
514/102 ;
435/006 |
International
Class: |
A61K 31/66 20060101
A61K031/66; C12Q 1/68 20060101 C12Q001/68 |
Claims
1-12. (canceled)
13. A method for identifying compounds that increase bone mineral
density, the method comprising contacting a compound with 15-LO and
determining whether the compound inhibits 15-LO.
14. The method of claim 13, further comprising testing the compound
in a functional assay that demonstrates an effect of the compound
on bone formation.
15. The method of claim 14, wherein the functional assay comprises
contacting the compound with human mesenchymal stem cells and
determining cellular differentiation into bone forming cells.
16. The method of claim 14, wherein the functional assay comprises
administering the compound to a non-human animal and measuring an
index of bone formation.
17. The method of claim 16, wherein the index measured is bone
mineral density.
18. The method of claim 16, wherein the index is a biomechanical
parameter of bone.
19. The method of claim 15, wherein determining cellular
differentiation comprises performing an alkaline phosphatase assay,
a calcium assay, a total DNA preparation assay, or combinations
thereof.
20. A method for identifying compounds that increase bone mineral
density, the method comprising contacting an inhibitor of
15-lipoxygenase with human mesenchymal stem cells and determining
cellular differentiation into bone forming cells.
21. The method of claim 20, wherein determining cellular
differentiation comprises performing an alkaline phosphatase assay,
a calcium assay, a total DNA preparation assay, or combinations
thereof.
22-28. (canceled)
Description
CROSS-REFERENCE TO RELATED INVENTION(S)
[0001] This application claims the priority benefit under Title 35
U.S.C. 119(e) of U.S. Provisional Application Ser. No. 60/355,255,
filed Feb. 8, 2002, the disclosure of which is herein incorporated
by reference in its entirety.
TECHNICAL FIELD
[0002] The invention relates generally to 15-lipoxygenase
inhibitors and their use in treating and 10 preventing bone loss.
Specifically, the invention relates to the use of 15-lipoxygenase
inhibitors to decrease bone loss and/or increase net bone
formation.
BACKGROUND OF THE INVENTION
[0003] Lipoxygenases are nonheme iron-containing enzymes found in
plants and animals that catalyze the oxygenation of certain
polyunsaturated fatty acids, such as lipids and lipoproteins.
Several different lipoxygenase enzymes are known, each having a
characteristic oxidation action. Mammalian lipoxygenases are named
by the position in arachidonic acid that is oxygenated. The enzyme
5-lipoxygenase converts arachidonic acid to
5-hydroperoxyeicosatetraenoic acid (5-HPETE). This is the first
step in the metabolic pathway which yields
5-hydroxyeicosatetraenoic acid (5-HETE) and the leukotrienes (LTs).
Similarly, 12- and 15-lipoxygenase convert arachidonic acid to 12-
and 15-HPETE, respectively. Biochemical reduction of 12-HPETE leads
to 12-HETE, while 15-HETE is the precursor of the class of
compounds known as lipoxins.
[0004] A diverse array of biological effects are associated with
the products of lipoxygenase activity, and many are implicated as
mediators in various disease states. The C4 and D4 LTs are potent
constrictors of human bronchial smooth muscle; LTB4 and 5-HETE,
found in the synovial fluid of patients with rheumatoid arthritis,
are potent chemotactic factors for inflammatory cells such as
polymorphonuclear leukocytes (Green and Lambeth, Tetrahedron, 39,
1687 (1983)); 12-HETE has been found at high levels in the
epidermal tissue of patients with psoriasis; the lipoxins have been
shown to stimulate lysosomal enzyme and superoxide ion release from
neutrophils. Thus, lipoxygenase enzymes play an important role in
the biosynthesis of mediators of asthma, allergy, arthritis,
psoriasis, and inflammation, and inhibitors of these enzymes
interrupt the biochemical pathway involved in these disease
states.
[0005] Human 15-lipoxygenase (15-LO) catalyzes the formation of
15-S-hydroxyeicosatetraenoic acid (15-S-HETE) from arachidonic acid
(Kuhn and Borngraber, Lipoxygenases and Their Metabolites, Plenum
Press, New York, (1999)). In mice, the synthesis of 15-S-HETE is
carried out by 12/15-Lipoxygenase (Alox 15), which is the murine
homologue of human 15-LO. Murine 12/15-LO converts arachidonic acid
to 12(S)-hydroxyeicosatetraenoic and 15-S-HETE in a 3:1 ratio, and
can additionally convert linoleic acid to
.beta.-hydroxyoctadecadienoic acid (13-HODE).
[0006] 15-Lipoxygenase has previously been implicated in the
pathogenesis of several diseases, including atherosclerosis (Harats
et al., Arterioscler. Thromb. Vasc. Biol., 2100-2105, (2000)),
asthma (Shannon et al., Am. Rev. Respir. Dis., 147, 1024-1028,
(1993)), cancer (Shureiqi et al., JNCI, 92, 1136-1142, (2000)), and
glomerulonephritis (Montero and Badr, Exp. Neph., 8, 14-19 (2000)).
A number of classes of compounds have been identified that inhibit
15-LO, including phenols, hydroxamic acids and acetylenic fatty
acids (reviewed in Kuhn and Borngraber, Lipoxygenases and Their
Metabolites, Plenum Press, New York, (1999)). The spectrum of
inhibitory activities varies for these agents. For example,
nordihydroguaiaretic acid has been shown to be an inhibitor of 5-
and 15-lipoxygenase, naphthylhydroxamic acids have been shown to
inhibit 5-, 12-, and 15-lipoxygenase (U.S. Pat. No. 4,605,669), and
a benzofluorene 15-LO inhibitor, PD146176, has been reported to be
relatively specific for the 15-LO enzyme (Sendobry et al., Br. J.
Pharm., 120, 1199-1206, (1997)). Evidence for 15-LO involvement in
atherosclerosis has come from studies with mice with a targeted
deletion of Alox 15 (Alox15 -/-). These Alox15 knockout mice were
initially shown to have minor phenotypic differences including
increased 5-LO activity (Sun and Funk, J. Biol. Chem., 271,
24055-24062, (1996)). However, disruption of Alox15 greatly
diminished atherosceloritic lesions in apoE deficient (apoE -/-)
atherosclerosis prone mice (Cyrus et al., J. Clin Invest., 103,
1597-1604, (1999)).
[0007] Although 5-LO has been implicated in the pathogenesis of
several diseases, the biologic function of murine or human 15-LO
has not been determined with certainty, nor has a human clinical
utility for inhibitors of 15-LO been established. In particular,
the utility of 15-LO inhibitors for treatment of human osteoporosis
and/or osteoarthritis has not previously been discovered.
[0008] Osteoporosis is caused by a reduction in bone mineral
density in mature bone and results in fractures after minimal
trauma. The disease is widespread and has a tremendous economic
impact. The most common fractures occur in the vertebrae, distal
radius and hip. An estimated one-third of the female population
over age 65 will have vertebral fractures, caused in part by
osteoporosis. Moreover, hip fractures are likely to occur in about
one in every three woman and one in every six men by extreme old
age.
[0009] Two distinct phases of bone loss have been identified. One
is a slow, age-related process that occurs in both genders and
begins at about age 35. This phase has a similar rate in both
genders and results in losses of similar amounts of cortical and
cancellous (spongy or lattice like) bone. Cortical bone
predominates in the appendicular skeleton while cancellous bone is
concentrated in the axial skeleton, particularly the vertebrae, as
well as in the ends of long bones. Osteoporosis caused by
age-related bone loss is known as Type II osteoporosis.
[0010] The other type of bone loss is accelerated, seen in
postmenopausal women and is caused by estrogen deficiency. This
phase results in a disproportionate loss of cancellous bone.
Osteoporosis due to estrogen depletion is known as Type I
osteoporosis. The main clinical manifestations of Type I
osteoporosis are vertebral, hip and forearm fractures. The skeletal
sites of these manifestations contain large amounts of trabecular
bone. Bone turnover is usually high in Type I osteoporosis. Bone
resorption is increased but there is inadequate compensatory bone
formation. Osteoporosis has also been related to corticosteroid
use, immobilization or extended bed rest, alcoholism, diabetes,
gonadotoxic chemotherapy, hyperprolactinemia, anorexia nervosa,
primary and secondary amenorrhea, transplant immunosuppression, and
oophorectomy.
[0011] The mechanism by which bone is lost in osteoporosis is
believed to involve an imbalance in the process by which the
skeleton renews itself. This process has been termed bone
remodeling. It occurs in a series of discrete pockets of activity.
These pockets appear spontaneously within the bone matrix on a
given bone surface as a site of bone resorption. Osteoclasts (bone
dissolving or resorbing cells) are responsible for the resorption
of a portion of bone of generally constant dimension. This
resorption process is followed by the appearance of osteoblasts
(bone forming cells) which then refill the cavity left by the
osteoclasts with new bone.
[0012] In a healthy adult subject, osteoclasts and osteoblasts
function so that bone formation and bone resorption are in balance.
However, in osteoporosis an imbalance in the bone remodeling
process develops which results in bone being replaced at a slower
rate than it is being lost. Although this imbalance occurs to some
extent in most individuals as they age, it is much more severe and
occurs at a younger age in postmenopausal osteoporosis, following
oophorectomy, or in iatrogenic situations such as those resulting
from the use of corticosteroids or immunosuppressants.
[0013] Various approaches have been suggested for increasing bone
mass in humans afflicted with osteoporosis, including
administration of androgens, fluoride salts, and parathyroid
hormone and modified versions of parathyroid hormone. It has also
been suggested that bisphosphonates, calcitonin, calcium,
1,25-dihydroxy vitamin D.sub.3, and/or estrogens, alone or in
combination, may be useful for preserving existing bone mass.
SUMMARY
[0014] The present invention is based on the discovery that
inhibitors of 15-lipoxygenase are able to increase net bone
formation and/or enhance bone accretion and enhance fracture
healing. These molecules can be delivered alone or in combination
with additional agents which inhibit bone resorption and/or enhance
bone formation, particularly anabolic agents
[0015] Accordingly, in one embodiment, the subject invention is
directed to a method for reducing bone loss in a subject. The
method comprises administering to the subject a pharmaceutically
effective amount of an inhibitor of 15-lipoxygenase.
[0016] In another embodiment, the invention is directed to a method
for increasing bone mineral density, which comprises administering
to the subject an effective amount of a pharmaceutically acceptable
inhibitor of 15-lipoxygenase.
[0017] In another embodiment, the invention is directed to a method
of diagnosis of predisposition to bone loss in a subject, the
method comprising detecting a polymorphism on human chromosome 17,
particularly detecting a polymorphism in the 15-LO gene.
[0018] In yet another embodiment, the invention is directed to a
method of screening for compounds for reducing bone loss or for
increasing bone mineral density by contacting inhibitors of
15-lipoxygenase with human mesenchymal stem cells and determining
cellular differentiation into cells involved in bone formation.
[0019] These and other aspects of the present invention will become
evident upon reference to the following detailed description and
attached figures. In addition, various references are set forth
herein which describe in more detail certain procedures or
compositions, and are therefore incorporated by reference in their
entirety.
BRIEF DESCRIPTION OF THE FIGURES
[0020] FIG. 1 depicts the comparison of SNP-based genotyping of
pooled DNA samples with microsatellite genotyping of individual DNA
samples for detecting polymorphisms related to bone mineral
density. The significance of each allele-frequency difference was
calculated using the z-test and plotted as an LOD score for all
chromosomes. Dashed line indicates an LOD score of 3.3 that was set
as the threshold for genome-wide significance.
[0021] FIG. 2 shows the quantitation of Alox15 gene expression in
mouse kidney using whole kidney RNA, where gene expression was
analyzed using microarrays.
[0022] FIG. 3 shows quantitation of Alox15 gene expression in mouse
primary osteoblasts in response to increasing concentrations of
IL-4 administered in vitro.
[0023] FIG. 4 shows the locations of the single nucleotide
polymorphisms (SNPs) identified in the Alox15 gene in mice.
[0024] FIG. 5 shows the quantitation of alkaline phosphatase
activity in human mesenchymal stem cells (hMSC) in response to
15-LO inhibitors Compound 1 or Compound 2 relative to solvent only
(DMSO) control.
[0025] FIG. 6 shows the quantitation of alkaline phosphatase
activity in hMSC with Compound 3 or Compound 2 as 15-LO inhibitors
and DMSO or 1,25-Vitamin D.sub.3 as the controls.
[0026] FIG. 7 shows the quantitation of the total calcium content
of hMSC with Compound 3 or Compound 2 as 15-LO inhibitors and DMSO
or 1,25-Vitamin D.sub.3 as the controls.
[0027] FIG. 8 shows the quantitation of alkaline phosphatase
activity in hMSC cultured for 9 days with the 5-LO inhibitors
Zileuton, AA-861, and Rev-5901 relative to solvent only (DMSO)
control. The lack of a response is in contrast to the effects seen
with inhibitors of 15-LO.
[0028] FIG. 9 shows the quantitation of the total calcium content
of hMSC cultured for 16 days with 5-LO inhibitors Zileuton, AA-861,
and Rev-5901 relative to solvent only (DMSO) or 1,25-Vitamin D3
controls. The lack of a response is in contrast to the effects seen
with inhibitors of 15-LO.
[0029] FIG. 10 shows the effect of 15-LO on bone density and
quality.
[0030] FIG. 11 shows that the correlation between the 15-LO
genotype and bone mineral density
[0031] FIG. 12 shows the correlation between 15-LO and bone
strength.
DETAILED DESCRIPTION OF THE INVENTION
[0032] The practice of the present invention will employ, unless
otherwise indicated, conventional methods of protein chemistry,
biochemistry, recombinant DNA techniques and pharmacology, within
the skill of the art. Such techniques are explained fully in the
literature.
[0033] See, e.g., T. E. Creighton, Proteins: Structures and
Molecular Properties (W.H. Freeman and Company, 1993); A. L.
Lehninger, Biochemistry (Worth Publishers, Inc., current addition);
Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd
Edition, 1989); Methods In Enzymology (S. Colowick and N. Kaplan
eds., Academic Press, Inc.); Remington's Pharmaceutical Sciences,
18th Edition (Easton, Pa.: Mack Publishing Company, 1990); Carey
and Sundberg Advanced Organic Chemistry 3.sup.rd Ed. (Plenum Press)
Vols A and B(1992).
[0034] All publications, patents and patent applications cited
herein, whether supra or infra, are hereby incorporated by
reference in their entirety.
I. Definitions
[0035] In describing the present invention, the following terms
will be employed, and are intended to be defined as indicated
below.
[0036] By "bone loss" is meant an imbalance in the ratio of bone
formation to bone resorption resulting in less bone than desirable
in a patient. Bone loss may result from osteoporosis, osteotomy,
periodontitis, or prosthetic loosening. Bone loss may also result
from secondary osteoporosis which includes glucocorticoid-induced
osteoporosis, hyperthyroidism-induced osteoporosis,
immobilization-induced osteoporosis, heparin-induced osteoporosis
or immunosuppressive-induced osteoporosis. Bone loss can be
monitored, for example, using bone mineral density measurements
described below.
[0037] The terms "effective amount" or "pharmaceutically effective
amount" refer to a nontoxic but sufficient amount of the agent to
provide the desired biological result. That result can be reduction
and/or alleviation of the signs, symptoms, or causes of a disease,
or any other desired alteration of a biological system. For
example, an "effective amount" for therapeutic uses is the amount
of the composition comprising an active compound herein required to
provide a clinically significant increase in healing rates in
fracture repair; reversal of bone loss in osteoporosis; reversal of
cartilage defects or disorders; prevention or delay of onset of
osteoporosis; stimulation and/or augmentation of bone formation in
fracture non-unions and distraction osteogenesis; increase and/or
acceleration of bone growth into prosthetic devices; and repair of
dental defects. An appropriate "effective" amount in any individual
case may be determined by one of ordinary skill in the art using
routine experimentation.
[0038] By "increased bone accretion" is meant that bone
accumulation in a subject administered the 15-lipoxygenase
inhibitors of the invention is increased over bone accumulation in
a comparable subject that is not given an 0.15-lipoxygenase
inhibitor. Such increased bone accretion is typically determined
herein by measuring bone mineral density (BMD). For example, bone
accretion can be determined using an animal model, such as an
ovariectomized mouse, dog and the like. The animal is administered
the test compound and bone mineral density (BMD) measured in bones
that are normally depleted in Type I or Type II osteoporosis, such
as bones of the appendicular and/or axial skeleton, particularly
the spine including the vertebrae, as well as in the ends of long
bones, such as the femur, midradius and distal radius. Several
methods for determining BMD are known in the art. For example, BMD
measurements may be done usingdual energy x-ray absorptiometry or
quantitative computed tomography, and the like. (See, the
examples.) Similarly, increased bone formation can be determined
using methods well known in the art. For example, dynamic
measurements of bone formation rate (BFR) can be performed on
tetracycline labeled cancellous bone from the lumbar spine and
distal femur metaphysis using quantitative digitized morphometry
(see, e.g., Ling et al., Endocrinology (1999) 140:5780-5788.
Alternatively, bone formation markers, such as alkaline phosphatase
activity and serum osteocalcin levels can be assessed to indirectly
determine whether increased bone formation has occurred (see Looker
et al., Osteoporosis International (2000) 11(6):467-480).
[0039] By "increased bone formation" is meant that the amount of
bone formation in a subject administered the 15-lipoxygenase
inhibitors of the invention is increased over the bone formation
rate in a subject that is not given an 15-lipoxygenase inhibitor.
Such enhanced bone formation is determined herein using, e.g.,
quantitative digitized morphometry, as well as by other markers of
bone formation, as described above.
[0040] By "inhibitor of 15-lipoxygenase" is meant a compound that
inhibits 15-LO with an IC50 of less than 1 .mu.M, preferably less
than 100 nM. IC50's may be determined by standard methods. One
particular method is a colorimetric assay in which the putative
inhibitor is pre-incubated with the 15-LO enzyme for about 10
minutes followed by addition of linoleic acid substrate for an
additional 10 minutes. The product, 13-HPODE, is quantitated by
coupling the reduction of the hydroperoxylated lipid to the
oxidation on N-benzoyl-leucomethylene blue in the presence of hemin
at pH 5. The absorbance of the oxidized methylene blue is directly
proportional to the amount of 13-HPODE formed by the 15-LO, and can
be measured in the presence and absence of inhibitor. This is an
endpoint assay described in more detail in "A Spectrophotometric
Microtiter-Based Assay for the Detection of Hydroperoxy Derivatives
of Linoleic Acid", Analytical Biochemistry, 201, 375-380 (1992);
Bruce J. Auerbach, John S. Kiely and Joseph A. Cornicelli. Other
assays include those where the initial enzyme reaction rate is
measured spectrophotometrically by measuring conjugated diene
formation at 234 nm.
[0041] As used herein, the terms "treat" or "treatment" are used
interchangeably and are meant to indicate a postponement of
development of bone loss symptoms and/or a reduction in the
severity of such symptoms that will or are expected to develop. The
terms further include preventing additional symptoms, ameliorating
or preventing the underlying metabolic causes of symptoms, and/or
encouraging bone growth.
[0042] By "pharmaceutically acceptable" or "pharmacologically
acceptable" is meant a material which is not biologically or
otherwise undesirable, i.e., the material may be administered to an
individual without causing any undesirable biological effects or
interacting in a deleterious manner with any of the components of
the composition in which it is contained.
[0043] By "physiological pH" or a "pH in the physiological range"
is meant a pH in the range of approximately 7.2 to 8.0 inclusive,
more typically in the range of approximately 7.2 to 7.6
inclusive.
[0044] As used herein, the term "subject" encompasses mammals and
non-mammals. Examples of mammals include, but are not limited to,
any member of the Mammalia class: humans, non-human primates such
as chimpanzees, and other apes and monkey species; farm animals
such as cattle, horses, sheep, goats, swine; domestic animals such
as rabbits, dogs, and cats; laboratory animals including rodents,
such as rats, mice and guinea pigs, and the like. Examples of
non-mammals include, but are not limited to, birds, fish and the
like. The term does not denote a particular age or gender.
[0045] As used herein, "polymorphism" refers to the occurrence of
two or more genetically determined alternative sequences or alleles
in a population. A polymorphic marker or site is the locus at which
divergence occurs. Preferred markers have at least two alleles,
each occurring at frequency of greater than 1%, and more preferably
greater than 10% or 20% of a selected population. A polymorphism
may comprise one or more base changes, an insertion, a repeat, or a
deletion. A polymorphic locus may be as small as one base pair.
Polymorphic markers include restriction fragment length
polymorphisms, variable number of tandem repeats (VNTR's),
hypervariable regions, minisatellites, dinucleotide repeats,
trinucleotide repeats, tetranucleotide repeats, simple sequence
repeats, and insertion elements.
[0046] A single nucleotide polymorphism (SNP) occurs at a
polymorphic site occupied by a single nucleotide, which is the site
of variation between allelic sequences. The site is usually
preceded by and followed by highly conserved sequences of the
allele (e.g., sequences that vary in less than 1/100 or 1/1000
members of the populations). A single nucleotide polymorphism
usually arises due to substitution of one nucleotide for another at
the polymorphic site. A transition is the replacement of one purine
by another purine or one pyrimidine by another pyrimidine. A
transversion is the replacement of a purine by a pyrimidine or vice
versa. Single nucleotide polymorphisms can also arise from a
deletion of a nucleotide or an insertion of a nucleotide relative
to a reference allele.
[0047] As used herein, the term "precursor cell" refers to a cell
that is not fully differentiated or committed to a differentiation
pathway, and that generally does not express markers or function as
a mature, fully differentiated cell.
[0048] As used herein, the term "mesenchymal cells" or "mesenchymal
stem cells" refers to pluripotent progenitor cells that are capable
of dividing many times, and whose progeny will give rise to
skeletal tissues, including cartilage, bone, tendon, ligament,
marrow stroma and connective tissue (see A. Caplan J. Orthop. Res.
(1991) 9:641-50).
[0049] As used herein, the term "osteogenic cells" includes
osteoblasts and osteoblast precursor cells.
[0050] As used herein, "Quantitative Trait Locus" (QTL) refers to a
phenotypic measure, such as bone mineral density, that is
continuously distributed and can be determined by multiple genes. A
QTL is a site on a chromosome whose alleles influence a
quantitative trait.
[0051] The compounds used in the present invention are those that
inhibit or reduce the activity of 15-lipoxygenase. In this context,
inhibition and reduction of the enzyme activity refers to a lower
level of measured activity relative to a control experiment in
which the enzyme, cell, or subject is not treated with the test
compound. In particular embodiments, the inhibition or reduction in
the measured activity is at least a 10% reduction or inhibition.
One of skill in the art will appreciate that reduction or
inhibition of the measured activity of at least 20%, 50%, 75%, 90%
or 100% or any integer between 10% and 100%, may be preferred for
particular applications. Typically, the 15-LO inhibitors used in
this invention will have IC50's of less than 1 .mu.M, preferably
less than 100 nM.
II. Identification and Validation of 15-LO
[0052] The present invention is based, in part, on the discovery
that 15-lipoxygenase is an important modulator of bone mass. In
particular, a genome scan of 100 microsatellite markers in mice led
to the identification of bone loss disorder susceptibility loci on
chromosomes 1, 2, 4, and 11. The differences in gene expression,
especially for genes encoded on chromosome 11, identified
substantial differences in expression of a gene encoding for
15-lipoxygenase, thereby linking the expression of 15-lipoxygenase
to a lowered bone mineral density.
[0053] Peak bone mass is a major determinant of osteoporotic
fracture risk. Family and twin studies have shown that genetic
factors regulate bone mineral density (BMD) (reviewed in Stewart
and Ralston, J. Endocr., (2000) 166: 235-245). The inventors herein
utilized a murine genetic model to identify loci that control BMD.
A two step method was used to identify the regions of the genome
that regulate bone mineral density. Initially, a computational
method and microsatellite markers were used to scan an animal
single nucleotide polymorphism (SNP) database, and the chromosomal
regions that contributed to acquisition and maintenance of skeletal
mass were predicted (Example 1). Subsequently, animal models were
used to identify and validate the mutations in the genes
contributing to the acquisition and maintenance of skeletal mass
(Example 2) and to show that disruption of the 15-LO gene causes
increased BMD (Example 4), thereby identifying 15-LO as a gene
involved in the regulation of bone mass.
III. Use of 15-Lipoxygenase Inhibitors
[0054] Compounds that inhibit 15-lipoxygenase activity and prevent
bone resorption or promote bone formation provide important
benefits to efforts at treating osteoporosis. Compounds that
inhibit 15-lipoxygenase activity can be used in a method for
treating osteoporosis or osteoarthritis by inhibition of
osteoclastic bone resorption or by stimulation of osteoblast
differentiation and promotion of new bone formation. Example 3
shows the ability of 15-LO inhibitors promote the differentiation
of human mesenchymal stem cells into osteoblasts in vitro. Example
4 shows that total disruption of the 15-LO gene in vivo leads to
increased BMD. Example 5 shows the ability of 15-LO inhibitors to
promote bone formation in an in vivo model.
[0055] Treatment with a 15-LO inhibitor may be used for healing of
bone fractures and osteotomies, including both union and nonunion
fractures. Types of fractures treatable by the methods of this
invention include both traumatic and osteoporotic fractures, e.g.,
fractures of the hip, neck of the femur, wrist, vertebrae, spine,
ribs, sternum, larynx and trachea, radius/ulna, tibia, patella,
clavicle, pelvis, humerus, lower leg, fingers and toes, face and
ankle. Both the rate of healing as well as promotion of union in a
fracture that would otherwise remain as a nonunion fracture may be
facilitated by the methods disclosed herein. Prophylactic treatment
of a patient identified to be at risk for fracture may also reduce
the fracture risk of that patient.
[0056] Several inhibitors of 15-lipoxygenase are known which may
find use with the subject methods. The inhibitors include synthetic
organic molecules, plant extracts and other natural products, and
antibodies against 15-LO. Representative and non-limiting examples
are described in Cornicelli J A, Trivedi B K. 15-lipoxygenase and
its inhibition: a novel therapeutic target for vascular disease.
[Review] [113 refs]. Current Pharmaceutical Design 1999; 5(1):
11-20 (describing various caffeic acid derivatives, propargyl
ethers, catechols and benzothiopyranoindoles); Cornicelli J A.
15-lipoxygenase inhibitors as antiatherosclerotic agents. IDrugs
1998; 1(2):206-213; Fleischer R, Frohberg P, Buge A, Nuhn P, Wiese
M. QSAR analysis of substituted 2-phenylhydrazonoacetamides acting
as inhibitors of 15-lipoxygenase. Quant Struct-Act Relat 2000;
19(2): 162-172; Kuhn H. Inhibitors of 12/15-lipoxygenase are
potential anti-atherosclerotic drugs. Curr Opin Anti-Inflammatory
Immunomodulatory Invest Drugs 1999; 1(3):227-237; Mogul R, Johansen
E, Holman T R. Oleyl sulfate reveals allosteric inhibition of
soybean lipoxygenase-1 and human 15-lipoxygenase. Biochemistry
2000; 39(16):4801-4807; Sexton K, Roark W H, Sorenson R, Cornicelli
J, Sekerke C, Welch K. Thiourea inhibitors of 15lipoxygenase.
Abstracts of Papers American Chemical Society 1999; 218(1-2):MEDI0;
Tait B D, Dyer R D, Auerbach B J, Bornemeier D, Guilds-Zamarka L,
Oxender M et al. Catechol based inhibitors of 15-lipoxygenase.
Bioorganic & Medicinal Chemistry Letters Vol 1996; 6(1):93-96;
Moreau R A, Agnew J, Hicks K B, Powell M J. Modulation of
lipoxygenase activity by bacterial hopanoids. Journal of Natural
Products Vol 1997; 60(4):397-398; 219th National Meeting of the
American Chemical Society (2000), Poster BIOL-15. Authors:
E-N-Jonsson and T-R-Holman. University of California, Santa Cruz,
Calif. (describing 15-LO inhibitors isolated from marine sponges;
and Lyckander IM, Malterud KE. Lipophilic flavonoids from
Orthosiphon spicatus as inhibitors of 15-lipoxygenase. Acta Pharm
Nord 1992; 4(3): 159-166. In addition, the thiourea and benzamide
compounds of U.S. Pat. No. 6,268,387 (incorporated by reference),
to Conner et al. (such as represented by
3-amino-N-(3,4-dichlorophenyl)-4-methoxy-benzamide Compound 3)
which are inhibitors of 15-lipoxygenase, as well as
2-phenyl-benzo[d]isoselenazol-3-one (ebselen),
6,11-dihydro-5-thia-11-aza-benzo[a]fluorine (termed Compound 2
herein), and phenyl acetylenic compounds represented by,
3-(2-oct-1-ynyl-phenyl)-acrylic acid Compound 4 are also useful in
the methods described herein. Also useful are compounds disclosed
in WO01/96298 (incorporated by reference), including Compound 6,
[[[5-(5,6-difluoro-1H-indol-2-yl)-2-methoxyphenyl]amino]sulfonyl]-carbami-
c acid-isobutyl ester.
[0057] Additional 15-LO inhibitors include those shown below;
##STR1##
[0058] described in 222nd National Meeting of the American Chemical
Society, Chicago, Ill., USA, 26-30 August, 2001. Poster, MEDI 270.
##STR2##
[0059] described in 218th ACS (New Orleans), 1999, MEDI 200.
[0060] 3) PD 146176 from Parke Davis (now Pfizer)
[0061] 4) A 78773 (Abbott) described in WO 92/1682 ##STR3##
[0062] described at 6th Int Conf Prostaglandins (Florence), 1986,
328
[0063] A related aspect of this invention relates to combination
therapies of 15-LO inhibitors for increased bone formation with
other active agents such as bisphosphonates, estrogen, SERMS
(selective estrogen receptor modulators), calcitonins or anabolic
therapies. Examples of bisphosphonates include alendronate,
ibandronate, pamidronate, etidronate and risedronate. Examples of
SERMS include raloxifene, dihydroraloxifene and lasofoxifene.
Calcitonins include human and salmon calcitonin. Anabolic agents
include parathyroid hormones (PTH) e.g. hPTH(1-34), PTH(1-84), and
parathyroid hormone-related protein (PTHrP) and analogs thereof.
Particular analogs of PTHrP are described in "Mono- and Bicyclic
Analogs of Parathyroid Hormone-Related Protein. 1. Synthesis and
Biological Studies," Michael Chorev et al. Biochemistry,
36:3293-3299 (1997) and "Cyclic analogs of PTH and PTHrP," WO
96/40193 and U.S. Pat. No. 5,589,452 and WO 97/07815. The other
active agent may be administered concurrently, prior to or after
the 15-LO inhibitor and may be administered by a different delivery
method. Preferably, the 15-LO inhibitor is administered first. The
period of this administration may be of any length, but typically
ranges from six to twenty four months. This treatment is then
followed by treatment with an antiresorptive agent, e.g., a
bisphosphonate, SERM, calcitonin or hormone replacement
therapy.
[0064] In one embodiment, a method of the present invention
involves the administration of a therapeutically effective amount
of an antisense oligonucleotide having a sequence capable of
binding specifically with any sequences of genomic DNA or an mRNA
molecule which encodes 15-lipoxygenase, so as to prevent
transcription or translation of 15-lipoxygenase mRNA. By
"antisense" is meant a composition containing a nucleic acid
sequence which is complementary to the "sense" strand of a specific
nucleic acid sequence. Once introduced into a cell, the
complementary nucleotides combine with endogenous sequences
produced by the cell to form duplexes and to block either
transcription or translation. See, e.g., Agrawal, S., ed. (1996)
Antisense Therapeutics, Humana Press Inc., Totawa N.J.; Alama et
al. (1997) Pharmacol. Res. 36:171-178; Crooke, S. T. (1997) Adv.
Pharmacol. 40:1-49; and Lavrosky et al. (1997) Biochem. Mol. Med.
62(1):11-22. Antisense sequences can be any nucleic acid material,
including DNA, RNA, or any nucleic acid mimics or analogs. See,
e.g., Rossi et al. (1991) Antisense Res. Dev. 1:285-288; Pardridge
et al. (1995) Proc. Nat. Acad. Sci. 92:5592-5596; Nielsen and
Haaima (1997) Chem. Soc. Rev. 96:73-78; and Lee et al. (1998)
Biochemistry 37:900-1010. Delivery of antisense sequences can be
accomplished in a variety of ways, such as through intracellular
delivery using a recombinant vector.
[0065] Antisense oligonucleotides of about 15 to 25 nucleic acid
bases are typically preferred as such are easily synthesized and
are capable of producing the desired inhibitory effect. Molecular
analogs of antisense oligonucleotides may also be used for this
purpose and can have added advantages such as stability,
distribution, or limited toxicity advantageous in a pharmaceutical
product. In addition, chemically reactive groups, such as
iron-linked ethylenediamine-tetraacetic acid (EDTA-Fe), can be
attached to antisense oligonucleotides, causing cleavage of the RNA
at the site of hybridization. These and other uses of antisense
methods to inhibit the in vitro translation of genes are well known
in the art. See, e.g., Marcus-Sakura (1988) Anal. Biochem.
172:289.
[0066] While a number of 15-lipoxygenase inhibitors are described
herein and known, it is understood that many others may be used in
the subject methods. The methods described herein are intended to
include the use of currently known 15-LO inhibitors as well as
those discovered subsequently. The assays described herein and
those known to one of skill in the art enable other 15-lipoxygenase
inhibitors that are useful for preventing or treating bone loss to
be readily identified and developed.
[0067] As shown in the Examples, genetic abnormalities or alleles
were found in chromosome 11, and the position of the allele
corresponded to the gene for 15-lipoxygenase. Thus, inhibition or
induction of 15-lipoxygenase has applications in various
situations. It is possible that by inhibiting 15-lipoxygenase
intracellularly, one may reduce bone turnover and thereby increase
bone mineral density and bone quality. Thus, inhibition of
15-lipoxygenase may be used in a method for treating or preventing
excessive resorption of bone, such as occurs in osteoporosis and
osteoarthritis.
[0068] The 15-lipoxygenase inhibitors of the present invention may
also be used to stimulate growth of bone-forming cells or their
precursors, or to induce differentiation of bone-forming cell
precursors, either in vitro or ex vivo. More particularly,
15-lipoxygenase inhibitors are useful for stimulating a cell
population containing marrow mesenchymal cells, thereby increasing
the number of osteogenic cells in that cell population. In a
preferred method, hematopoietic cells are removed from the cell
population, either before or after treatment with 15-lipoxygenase
inhibitors. Through practice of such methods, osteogenic cells may
be expanded. The expanded osteogenic cells can be infused (or
reinfused) into a vertebrate subject in need thereof. For instance,
a subject's own mesenchymal stem cells can be exposed to compounds
of the present invention ex vivo, and the resultant osteogenic
cells could be infused or directed to a desired site within the
subject, where further proliferation and/or differentiation of the
osteogenic cells can occur without immunorejection. Alternatively,
the cell population exposed to the 15-lipoxygenase inhibitors may
be immortalized human fetal osteoblastic or osteogenic cells. If
such cells are infused or implanted in a vertebrate subject, it may
be advantageous to immunoprotect these non-self cells, or to
immunosuppress (preferably locally) the recipient to enhance
transplantation and bone or cartilage repair.
IV. Methods for Identifying Agents for Treating or Preventing Bone
Loss
[0069] Several methods for identifying classes of 15-lipoxygenase
inhibitors that may also prevent bone loss and/or promote bone
formation may be employed. One method used to identify compounds
that inhibit 15-lipoxygenase activity involves placing cells,
tissues, or preferably a cellular extract or other preparation
containing 15-lipoxygenase in contact with several known
concentrations of a test compound in a buffer compatible with
15-lipoxygenase activity. The level of 15-lipoxygenase activity for
each concentration of test compound is measured by quantitation of
enzyme product and the IC.sub.50 (the concentration of the test
compound at which the observed activity for a sample preparation is
observed to fall one-half of its original or a control value) for
the compound is determined using standard techniques. Other methods
for determining the inhibitory concentration of a compound of the
invention against 15-lipoxygenase are known to one of skill in the
art and can be employed as will be apparent based on the disclosure
herein. Specific assays that may be used to identify 15-LO
inhibitors include those described in U.S. Pat. Nos. 6,268,387B1
and 5,958,950 (rabbit reticulocyte assay) and U.S. Pat. No.
4,623,661 (radiolabelled arachidonic acid in RBL-1 cells)
[0070] For example, antagonists can normally be found once a ligand
has been structurally defined. Testing of potential ligand analogs
is now possible upon the development of highly automated assay
methods using physiologically responsive cells. In particular, new
agonists and antagonists will be discovered by using screening
techniques described herein.
[0071] In another method, rational drug design, based upon
structural studies of the molecular shapes of the chemokines, other
effectors or analogs, or the receptors, may be used to identifying
compounds whose three-dimensional structure is complementary to
that of the active site of 15-lipoxygenase. These compounds may be
determined by a variety of techniques including molecular mechanics
calculations, molecular dynamics calculations, constrained
molecular dynamics calculations in which the constraints are
determined by NMR spectroscopy, distance geometry in which the
distance matrix is partially determined by NMR spectroscopy, x-ray
diffraction, or neutron diffraction techniques. In the case of all
these techniques, the structure can be determined in the presence
or absence of any ligands known to interact with
15-lipoxygenase.
[0072] Such computer programs include but are not limited to AMBER
(available from University of California, San Francisco), CHARMM
(Chemistry at HARvard Molecular Mechanics, available from Harvard
University), MM2, SYBYL (Trypos Inc.), CHEMX (Chemical Design),
MACROMODEL, GRID (Molecular Discovery Ltd), and Insight II
(Accelryl). Such programs are contemplated as being useful for the
determination of the chemical interaction between two molecules,
either isolated, or surrounded by solvent molecules, such as water
molecules, or using calculations that approximate the effect of
solvating the interacting molecules. The relative orientation of
the two can be determined manually, by visual inspection, or by
using other computer programs which generate a large number of
possible orientations. Examples of computer programs include but
are not limited to DOCK and AutoDOCK. Each orientation can be
tested for its degree of complementarity using the computer
programs. Thus, novel compounds can be designed that are capable of
inhibiting 15-lipoxygenase.
[0073] Another method for identifying compounds that may inhibit
15-lipoxygenase involves the use of techniques such as UV/VIS
spectroscopy, polarimetry, CD or ORD spectroscopy, IR or Raman
spectroscopy, NMR spectroscopy, fluorescence spectroscopy, HPLC,
gel electrophoresis, capillary gel electrophoresis, dialysis,
refractometry, conductometry, atomic force microscopy,
polarography, dielectometry, calorimetry, solubility, EPR or mass
spectroscopy. The application of these methods can be direct, in
which the compound's interaction with 15-lipoxygenase is measured
directly, or it can be indirect, in which a particular agent having
a useful spectroscopic property is used as a probe for the ability
of other compounds to bind to 15-lipoxygenase; for example, by
displacement or by fluorescence quenching.
[0074] The 15-lipoxygenase inhibitors thus identified or designed
can be subsequently tested for their ability to prevent bone loss
and/or promote bone formation. In one embodiment, the computer
based methods discussed above are used. In another method, the
compounds are tested for their ability to modulate known targets
for bone loss, such as, for example, estrogen receptors, tumor
necrosis factor receptor, integrin receptor, and the like. In yet
another method, the compounds are tested for their ability to
differentiate stem cells into bone cells.
V. Pharmaceutical Formulations and Modes of Administration
[0075] The methods described herein use pharmaceutical compositions
comprising the molecules described above, together with one or more
pharmaceutically acceptable excipients or vehicles, and optionally
other therapeutic and/or prophylactic ingredients. Such excipients
include liquids such as water, saline, glycerol,
polyethyleneglycol, hyaluronic acid, ethanol, etc. Suitable
excipients for nonliquid formulations are also known to those of
skill in the art. Pharmaceutically acceptable salts can be used in
the compositions of the present invention and include, for example,
mineral acid salts such as hydrochlorides, hydrobromides,
phosphates, sulfates, and the like; and the salts of organic acids
such as acetates, propionates, malonates, benzoates, and the like.
A thorough discussion of pharmaceutically acceptable excipients and
salts is available in Remington's Pharmaceutical Sciences, 18th
Edition (Easton, Pa.: Mack Publishing Company, 1990).
[0076] Additionally, auxiliary substances, such as wetting or
emulsifying agents, biological buffering substances, surfactants,
and the like, may be present in such vehicles. A biological buffer
can be virtually any solution which is pharmacologically acceptable
and which provides the formulation with the desired pH, i.e., a pH
in the physiologically acceptable range. Examples of buffer
solutions include saline, phosphate buffered saline, Tris buffered
saline, Hank's buffered saline, and the like.
[0077] Depending on the intended mode of administration, the
pharmaceutical compositions may be in the form of solid, semi-solid
or liquid dosage forms, such as, for example, tablets,
suppositories, pills, capsules, powders, liquids, suspensions,
creams, ointments, lotions or the like, preferably in unit dosage
form suitable for single administration of a precise dosage. The
compositions will include an effective amount of the selected drug
in combination with a pharmaceutically acceptable carrier and, in
addition, may include other pharmaceutical agents, adjuvants,
diluents, buffers, etc.
[0078] For solid compositions, conventional nontoxic solid carriers
include, for example, pharmaceutical grades of mannitol, lactose,
starch, magnesium stearate, sodium saccharin, talc, cellulose,
glucose, sucrose, magnesium carbonate, and the like. Liquid
pharmaceutically administrable compositions can, for example, be
prepared by dissolving, dispersing, etc., an active compound as
described herein and optional pharmaceutical adjuvants in an
excipient, such as, for example, water, saline, aqueous dextrose,
glycerol, ethanol, and the like, to thereby form a solution or
suspension. If desired, the pharmaceutical composition to be
administered may also contain minor amounts of nontoxic auxiliary
substances such as wetting or emulsifying agents, pH buffering
agents and the like, for example, sodium acetate, sorbitan
monolaurate, triethanolamine sodium acetate, triethanolamine
oleate, etc. Actual methods of preparing such dosage forms are
known, or will be apparent, to those skilled in this art; for
example, see Remington's Pharmaceutical Sciences, referenced
above.
[0079] For oral administration, the composition will generally take
the form of a tablet, capsule, a softgel capsule or may be an
aqueous or nonaqueous solution, suspension or syrup. Tablets and
capsules are preferred oral administration forms. Tablets and
capsules for oral use will generally include one or more commonly
used carriers such as lactose and corn starch. Lubricating agents,
such as magnesium stearate, are also typically added. When liquid
suspensions are used, the active agent may be combined with
emulsifying and suspending agents. If desired, flavoring, coloring
and/or sweetening agents may be added as well. Other optional
components for incorporation into an oral formulation herein
include, but are not limited to, preservatives, suspending agents,
thickening agents, and the like.
[0080] Parenteral formulations can be prepared in conventional
forms, either as liquid solutions or suspensions, solid forms
suitable for solubilization or suspension in liquid prior to
injection, or as emulsions. Preferably, sterile injectable
suspensions are formulated according to techniques known in the art
using suitable carriers, dispersing or wetting agents and
suspending agents. The sterile injectable formulation may also be a
sterile injectable solution or a suspension in a nontoxic
parenterally acceptable diluent or solvent. Among the acceptable
vehicles and solvents that may be employed are water, Ringer's
solution and isotonic sodium chloride solution. In addition,
sterile, fixed oils, fatty esters or polyols are conventionally
employed as solvents or suspending media. In addition, parenteral
administration may involve the use of a slow release or sustained
release system such that a constant level of dosage is
maintained.
[0081] Alternatively, the pharmaceutical compositions of the
invention may be administered in the form of suppositories for
rectal administration. These can be prepared by mixing the agent
with a suitable nonirritating excipient which is solid at room
temperature but liquid at the rectal temperature and therefore will
melt in the rectum to release the drug. Such materials include
cocoa butter, beeswax and polyethylene glycols.
[0082] The pharmaceutical compositions of the invention may also be
administered by nasal aerosol or inhalation. Such compositions are
prepared according to techniques well-known in the art of
pharmaceutical formulation and may be prepared as solutions in
saline, employing benzyl alcohol or other suitable preservatives,
absorption promoters to enhance bioavailability, propellants such
as fluorocarbons or nitrogen, and/or other conventional
solubilizing or dispersing agents.
[0083] Preferred formulations for topical drug delivery are
ointments and creams. Ointments are semisolid preparations which
are typically based on petrolatum or other petroleum
derivatives.
[0084] Creams containing the selected active agent, are, as known
in the art, viscous liquid or semisolid emulsions, either
oil-in-water or water-in-oil. Cream bases are water-washable, and
contain an oil phase, an emulsifier and an aqueous phase. The oil
phase, also sometimes called the "internal" phase, is generally
comprised of petrolatum and a fatty alcohol such as cetyl or
stearyl alcohol; the aqueous phase usually, although not
necessarily, exceeds the oil phase in volume, and generally
contains a humectant. The emulsifier in a cream formulation is
generally a nonionic, anionic, cationic or amphoteric surfactant.
The specific ointment or cream base to be used, as will be
appreciated by those skilled in the art, is one that will provide
for optimum drug delivery. As with other carriers or vehicles, an
ointment base should be inert, stable, nonirritating and
nonsensitizing.
[0085] Formulations for buccal administration include tablets,
lozenges, gels and the like. Alternatively, buccal administration
can be effected using a transmucosal delivery system as known to
those skilled in the art. The compounds of the invention may also
be delivered through the skin or muscosal tissue using conventional
transdermal drug delivery systems, i.e., transdermal "patches"
wherein the agent is typically contained within a laminated
structure that serves as a drug delivery device to be affixed to
the body surface. In such a structure, the drug composition is
typically contained in a layer, or "reservoir," underlying an upper
backing layer. The laminated device may contain a single reservoir,
or it may contain multiple reservoirs. In one embodiment, the
reservoir comprises a polymeric matrix of a pharmaceutically
acceptable contact adhesive material that serves to affix the
system to the skin during drug delivery. Examples of suitable skin
contact adhesive materials include, but are not limited to,
polyethylenes, polysiloxanes, polyisobutylenes, polyacrylates,
polyurethanes, and the like. Alternatively, the drug-containing
reservoir and skin contact adhesive are present as separate and
distinct layers, with the adhesive underlying the reservoir which,
in this case, may be either a polymeric matrix as described above,
or it may be a liquid or gel reservoir, or may take some other
form. The backing layer in these laminates, which serves as the
upper surface of the device, functions as the primary structural
element of the laminated structure and provides the device with
much of its flexibility. The material selected for the backing
layer should be substantially impermeable to the active agent and
any other materials that are present.
[0086] A pharmaceutically or therapeutically effective amount of
the composition will be delivered to the subject. The precise
effective amount will vary from subject to subject and will depend
upon the species, age, the subject's size and health, the nature
and extent of the condition being treated, recommendations of the
treating physician, and the therapeutics or combination of
therapeutics selected for administration. Thus, the effective
amount for a given situation can be determined by routine
experimentation. For purposes of the present invention, generally a
therapeutic amount will be in the range of about 0.05 mg/kg to
about 40 mg/kg body weight, more preferably about 0.5 mg/kg to
about 20 mg/kg, in at least one dose. In larger mammals the
indicated daily dosage can be from about 1 mg to 100 mg, one or
more times per day, more preferably in the range of about 10 mg to
50 mg. The subject may be administered as many doses as is required
to reduce and/or alleviate the signs, symptoms, or causes of the
disorder in question, or bring about any other desired alteration
of a biological system.
[0087] The delivery of polynucleotides, e.g., for delivering
15-lipoxygenase antisense oligonucleotides, can be achieved using
any of the formulations described above, or by using recombinant
expression vectors, with or without carrier viruses or particles.
Such methods are well known in the art. See, e.g., U.S. Pat. Nos.
6,214,804; 6,147,055; 5,703,055; 5,589,466; 5,580,859; Slater et
al. (1998) J. Allergy Clin. Immunol. 102:469-475. For example,
delivery of polynucleotide sequences can be achieved using various
viral vectors, including retrovirus and adeno-associated virus
vectors. See, e.g., Miller A. D. (1990) Blood 76:271; and Uckert
and Walther (1994) Pharmacol. Ther. 63:323-347. Vectors which can
be utilized for antisense gene therapy include, but are not limited
to, adenoviruses, herpes viruses, vaccinia, or, preferably, RNA
viruses such as retroviruses. Other gene delivery mechanisms that
can be used for delivery of polynucleotide sequences to target
cells include colloidal dispersion and liposome-derived systems,
artificial viral envelopes, and other systems available to one of
skill in the art. See, e.g., Rossi, J. J. (1995) Br. Med. Bull.
51:217-225; Morris et al. (1997) Nucl. Acids Res. 25:2730-2736; and
Boado et al. (1998) J. Pharm. Sci. 87:1308-1315. For example,
delivery systems can make use of macromolecule complexes,
nanocapsules, microspheres, beads, and lipid-based systems
including oil-in-water emulsions, micelles, mixed micelles, and
liposomes.
[0088] As discussed above, the pharmaceutical formulations may
contain one or more active agents that effectively regulate calcium
homeostasis. The additional active agent may be, but is not limited
to, an estrogen, a calcitonin, a bisphosphonate (e.g. alendronate,
residronate, zolendronate and ibandronate), vitamin D.sub.3 or an
analogue thereof, an androgen, a fluoride salt, a parathyroid
hormone or an analogue thereof, or an IGF, agents that alter
regulation of transcription of naturally occurring hormone
regulators involved in bone metabolism, and combinations thereof.
This additional active agent can be administered to the subject
prior to, concurrently with or subsequently to administration of
the 15-lipoxygenase inhibitor of this invention.
VI. Diagnosis and Predisposition
[0089] Another aspect of the present invention is based upon the
discovery of a correlation between a polymorphism in a
15-lipoxygenase gene and bone mineral density wherein the presence
of certain polymorphisms correlates with decreased bone mineral
density. A further aspect of the discovery is that the genotype is
correlated with a predisposition to osteoporosis. The invention is
of advantage in that by screening for the presence of the genotype
it is possible to identify individuals likely to have this genetic
predisposition. Accordingly, a further aspect of the invention
provides a method of therapy comprising screening an individual for
a predisposition to bone loss and, if a predisposition is
identified, treating that individual to delay or reduce or prevent
bone loss.
[0090] According to the diagnostic and prognostic method of the
present invention, alteration, including deletions, insertions and
point mutations in the coding and noncoding regions, of the
wild-type 15-lipoxygenase locus is detected. Thus, in mice, for
example, alternations on chromosome 11 are preferably detected,
whereas in humans, alternations on chromosome 17 are detected. In
addition, the method can be performed by detecting the wild-type
15-lipoxygenase locus and confirming the lack of a predisposition
to bone loss disorders at the 15-lipoxygenase locus. Such mutations
may be present in individuals either with or without symptoms of
bone loss. In addition, there may be differences in the drug
response or prognosis of symptomatic individuals that carry
mutations in 15-lipoxygenase locus compared to those that do
not.
[0091] Accordingly, one aspect of the invention provides a method
of diagnosis comprising determining the genotype of the
15-lipoxygenase gene. Typically, the method determines whether an
individual is homozygous or heterozygous for 15-lipoxygenase gene
polymorphisms. Typically, the method of the invention is carried
out by in vitro analysis of cells of an individual to determine the
genotype of that individual at the 15-lipoxygenase gene locus.
[0092] Useful diagnostic techniques include, but are not limited
to, microarray analysis, fluorescent in situ hybridization (FISH),
direct DNA sequencing, PFGE analysis, Southern blot analysis,
single stranded conformation analysis (SSCA), RNase protection
assay, allele-specific oligonucleotide (ASO) analysis, dot blot
analysis and PCR-SSCP, as is well known in the art.
[0093] Predisposition to bone loss disease can be ascertained by
testing any tissue of a subject, such as a human patient, for
mutations of the 15-lipoxygenase gene. For example, a person who
has inherited a germline 15-lipoxygenase mutation would be prone to
develop bone loss disease. This can be determined by testing DNA
from any tissue of the person's body. Most simply, blood can be
drawn and DNA extracted from the cells of the blood. In addition,
prenatal diagnosis can be accomplished by testing fetal cells,
placental cells or amniotic cells for mutations of the
15-lipoxygenase gene. Alteration of a wild-type 15-lipoxygenase
allele, whether, for example, by point mutation or deletion, can be
detected by any of the means discussed herein.
[0094] There are several methods that can be used to detect DNA
sequence variation. Direct DNA sequencing, either manual sequencing
or automated fluorescent sequencing can detect sequence variation.
Another approach is the single-stranded conformation polymorphism
assay (SSCA), where the fragments with shifted mobility on SSCA
gels are sequenced to determine the exact nature of the DNA
sequence variation. Other approaches based on the detection of
mismatches between the two complementary DNA strands include
clamped denaturing gel electrophoresis (CDGE), heteroduplex
analysis (HA) and chemical mismatch cleavage (CMC). Once a mutation
is known, an allele specific detection approach such as allele
specific oligonucleotide (ASO) hybridization can be utilized to
rapidly screen large numbers of other samples for that same
mutation (see, e.g., Saiki et al., Proc. Nad. Acad. Sci. USA
86:6230-6234 (1989)).
[0095] Detection of point mutations may be accomplished by
molecular cloning of the 15-lipoxygenase allele(s) and sequencing
the allele(s) using techniques well known in the art.
Alternatively, the gene sequences can be amplified directly from a
genomic DNA preparation from the tissue, using known techniques.
The DNA sequence of the amplified sequences can then be determined.
The presence of an allele can be confirmed by single-stranded
conformation analysis (SSCA), denaturing gradient gel
electrophoresis (DGGE or CDGE), RNase protection assays,
allele-specific oligonucleotides (ASOs), allele-specific PCR, or
single nucleotide extension assays, as is well known in the
art.
[0096] In the preferred method, alleles are identified by using
microchip technology. In this technique, thousands of distinct
oligonucleotide probes or genes can be synthesized (U.S. Pat. No.
5,412,087 to McGall et al.) or spotted as an array (U.S. Pat. No.
6,110,426 to Shalon et al.) on a silicon or glass chip. Nucleic
acid to be analyzed is generally fluorescently labeled and
hybridized to the probes on the chip. Other labels include .sup.32P
and biotin. It is also possible to study nucleic acid-protein
interactions using these microarrays. Using this technique, the
presence of mutations in the 15-lipoxygenase gene is
identified.
[0097] The presence of an altered (or a mutant) 15-lipoxygenase
gene correlates to an increased risk of bone loss disease. In order
to detect a 15-lipoxygenase gene mutation, a biological sample is
prepared and analyzed for a difference between the sequence of the
15-lipoxygenase allele being analyzed and the sequence of the
wild-type 15-lipoxygenase allele. The mutant alleles can then be
sequenced to identify the specific mutation of the particular
mutant allele. The mutations in the 15-lipoxygenase gene,
specifically on chromosome 11 for mice and chromosome 17 for
humans, are then used for the diagnostic and prognostic methods of
the present invention.
VII. EXPERIMENTAL
[0098] Below are examples of specific embodiments for carrying out
the present invention. The examples are offered for illustrative
purposes only, and are not intended to limit the scope of the
present invention in any way. TABLE-US-00001 Cpd. No. Structure
IC50 for 15-LO 1 ##STR4## 1-3 .mu.M 2 ##STR5## .about.200 nM 3
##STR6## .about.10 nM 4 ##STR7## 1-3 .mu.M 5 ##STR8## negative
control 6 ##STR9## 25-83 nM
[0099] Compound 1, 3-(2-non-1-ynylphenyl)-propionic acid, is
described in U.S. Pat. No. 5,972,980.
[0100] Compound 2, 6,11-dihydro-5-thia-11-aza-benzo[a]fluorene, is
described in WO97/12613.
[0101] Compound 3,
3-amino-N-(3,4-dichlorophenyl)-4-methoxybenzamide, is described in
WO99/32433.
[0102] Compound 4, trans-3-(2-oct-1-ynyl-phenyl)-acrylic acid is
described in U.S. Pat. No. 4,713,486.
[0103] Compound 5, Zilueton, is described in U.S. Pat. No.
4,873,259.
[0104] Compound 6,
[[[5-(5,6-difluoro-1H-indol-2-yl)-2-methoxyphenyl]amino]sulfonyl]-carbami-
c acid-isobutyl ester, is described in WO01/96298.
[0105] Efforts have been made to ensure accuracy with respect to
numbers used (e.g., amounts, temperatures, etc.), but some
experimental error and deviation should, of course, be allowed
for.
Example 1
SNP Genotyping Models
[0106] Microsatellites (also called simple tandem repeat
polymorphisms, or simple sequence length polymorphisms) constitute
the most developed category of genetic markers. They include small
arrays of tandem repeats of simple sequences
(di-,tri-,tetra-nucleotide repeats) which exhibit a high degree of
length polymorphism thereby providing a high level of information.
Slightly more than 5,000 microsatellites easily typed by
PCR-derived technologies, have been ordered along the human genome
(Dib et al., Nature 380:152 (1996)).
[0107] The method described by Grupe et al. (2001) Science 292:
1915-1918 was generally followed. To identify genetic factors
regulating BMD, a genome scan was performed on 1000 F2 progeny of a
C57BL/6.times.DBA/2 intercross (Jackson Labs, Bar Harbor, Me.) at
16 weeks of age. The F2 progeny displayed a non-sex linked, normal
distribution of BMD (Grupe et al., Science, 292, 1915-1918,
(2001)). Phenotypically extreme F2 progeny with the highest (n=145)
and lowest (n=149) BMD (top and bottom 15%) were subjected to a
whole-genome-scan for association with BMD by genotyping individual
DNA samples with 100 microsatellite markers. In addition, equal
amounts of DNA from the high and low BMD F2 progeny were used to
form two pools. Allele frequencies in the pooled samples were
measured for 109 SNPs found in the mSNP database using the
allele-specific kinetic PCR method. Differences in allele-frequency
between the two extremes for each marker were scored. If a marker
has no association with BMD, its expected frequency is 50% for both
extremes. The significance of each allele-frequency difference was
calculated using the z-test and plotted as a LOD score (FIG. 1). A
significant association (LOD score >3.3) was found for four
regions, located on chromosomes 1, 2, 4, and 11 by the
microsatellite and SNP genotyping methods.
[0108] Thus, using the SNP genotyping method, candidate genes
responsible for BMD were identified.
Example 2
Animal Models
[0109] To identify the gene within the identified region on
chromosome 11 which regulated BMD, gene expression differences
between DBA/2, C57BL6/J (Jackson Labs, Bar Harbor, Me.) and a
congenic mouse strain (D2.B6chr11, obtained from Oregon Health
Sciences University) were analyzed using microarrays. The
D2.B6chr11 congenic mouse had the C57BL/6J chromosome 11 interval
from cM20 to cM50 introgressed onto the DBA2/J background.
Microarrays, containing genes from the mouse genome, were
hybridized with labeled cRNA obtained from whole kidneys, cultured
primary osteoblasts and primary chondrocytes. Hybridizations were
done with cRNA obtained from individual DBA/2J, C57BL/6J, and
D2.B6chr11 congenic mice. Isolation of mRNA (2.times. poly(A)+),
cRNA synthesis and hybridization were performed. Differential
expression was assessed by pairwise comparison of DBA/2J and
C57BL/6 mice, and by pairwise comparison of DBA/2J and D2.B6chr11
congenic mice.
[0110] Differentially expressed genes, encoded within the interval
(cM20-cM50) on chromosome 11 were selected for further
characterization. A single gene (Alox 15 at cM 40 on chr. 11) was
substantially differentially expressed between the two strains
examined (FIG. 2). The DBA/2J strain had elevated levels of
expression of Alox15 compared to D2.B6chr11 and C57BL/6J mice and
also decreased BMD relative to both the other strains. Thus,
elevated Alox15 expression in the DBA/2J mice contributed to a
lowered bone mineral density.
[0111] Differential Alox15 mRNA expression in osteoblast cells was
assessed using real-time PCR. The cells were prepared from DBA/2J,
C57BL/6J, and D2.B6chr11 mice (FIG. 3). Total RNA was treated with
DnaseI to remove contaminating genomic DNA. The RNA was heated at
65.degree. C. for 10 min to inactivate the DnaseI. All PCR
reactions were performed in a 100-.mu.l volume. The RNA (100 ng)
was subjected to RT PCR using rTh DNA polymerase (2 units) (Perkin
Elmer) in a reaction mix containing 5.times.EZ buffer, Mn(OAc) (3
mM), ethidium bromide (1 .mu.g/ul), dNTPs (200 .mu.M), forward
primer (200 nM), and reverse primer (200 nM). The cDNA was
generated by incubating samples for 30 min at 60.degree. C. This
was followed by PCR for 60 cycles (95.degree. C., 20 sec;
58.degree. C., 20 sec), and then incubated for 20 min at 72.degree.
C. The fluorescence at each cycle is related to the amount of
product generated and is measured using a kinetic thermocycler and
analyzed using software provided (Germer and Higuchi, Genome
Research, 9, 72-78, (1999)). In addition to the kidney, osteoblasts
from DBA/2J mice also produced elevated basal levels of Alox15 m
RNA compared to C57BL/6J mice. Alox15 RNA was induced by IL-4 in
the osteoblasts from C57BL/6 mice to a greater degree relative to
DBA/2 mice (FIG. 3).
[0112] To identify a molecular basis for the strain-specific
difference in ALox15 expression, the genomic DNA for the Alox15
gene from DBA/2J and C57BL6/J mice was sequenced using methods
known in the art. Fourteen DNA sequence polymorphisms
distinguishing the two strains were identified (diagramed in FIG. 4
and in Table 1 below). TABLE-US-00002 TABLE 1 Alox15 SNPs
identified in DBA/2J compared to C57BL/6J mice. Nucleotide Change
SNP Position (bp) C57BL/6J_DBA2/J Intron/Exon A.A. Position 541 of
U04332 C_G .about.1 kb upstream of putative TATA box in 5' utr
.about.156 of Intron 1 A G Intron 1 .about.361 of Intron 1 T_C
Intron 1 .about.363 of Intron 1 A T Intron 1 .about.418 of Intron 1
C_T Intron 1 186 of U04331 T_C Exon 2 62/Phe(TTT)_Phe(TTC) 273 of
U04331 G_A Exon 2 91/Ser(TCG)_Ser(TCA) .about.50 of Intron 2 G_A
Intron 2 .about.10 of Intron 3 G A Intron 3 .about.20 of Intron 3
C_T Intron 3 .about.110 of Intron 10 A G Intron 10 .about.25 of
Intron 13 A_G Intron 13 1846 of U04331 C_T Exon 14
616/Pro(CCA)_Ser(TCA) 1851 of U04331 C_T Exon 14
617/Asn(AAC)_Asn(AAT) 2013 of L34570 T deleted in DBA/2J 11th base
of 3' utr
Example 3
Ability of 15-lipoxygenase Inhibitors to Increase Bone
Formation
[0113] In order to determine the ability of inhibitors of
15-lipoxygenase to stimulate bone formation and bone accretion, the
ability of the 15-LO inhibitors to promote measures of
differentiation of stem cells into bone forming cells (osteoblasts)
was tested in vitro. In particular, the ability of three
inhibitors, Compound 1 (3-(2-non-1-ynylphenyl)-propionic acid),
Compound 2 (6,11-dihydro-5-thia-11-aza-benzo[a]fluorene), and
Compound 3 (3-amino-N-(3,4-dichlorophenyl)-4-methoxybenzamide),
prepared using literature procedures, to promote the
differentiation of human mesenchymal stem cells into osteoblasts
was determined by measuring two markers of osteoblast
differentiation, cellular alkaline phosphatase activity and culture
calcium content.
[0114] In general, human mesenchymal stem cells (hMSC, Poietics
#PT-2501, BioWhittaker) were cultured in 12-well collagen coated
culture plates at 3100 cells per square cm in 1 ml culture medium
(Clonetics cat#PT-4105 plus osteoblast inducers (100 nM Dex, 0.05
mM L-Ascorbic acid-2-phosphate, 10 mM beta-glycerolphosphate). The
three 15-LO inhibitors were added individually to each of the
cultures, except for control wells to which was added 0.1% DMSO
(negative control) or 1 nM of 1,25-Vitamin D.sub.3 (positive
control). The concentrations of the inhibitors were as follows:
Compound 1 at 3 and 30 .mu.M; Compound 2 at 0.1, 0.2, 0.3, or 1.0
.mu.M and Compound 3 at 3, 10, or 30 .mu.M. Four to eight
independent replicate cultures were prepared at each dose level. At
the end of the experiments, the cultured cells were harvested by
scraping the wells into the appropriate medium for further analysis
of either alkaline phosphatase (Sigma kit#104-LL), or cellular
calcium (Sigma kit#587-M). Cultures harvested for alkaline
phosphatase assessment were harvested by scraping the cells and
resuspending into 250 .mu.L Tris buffered 0.1% Triton X-100, and
then assayed either fresh or following freeze-thaw. Separate
cultures to be assayed for total calcium content were scraped and
resuspended into 250 .mu.L 0.5M HCL. Results of these assays were
normalized to total cellular DNA, thereby normalizing for
differences in cell proliferation. At the same times that cultures
were harvested for alkaline phosphatase and calcium, separate
replicate cultures were harvested by scraping the cells and
resuspending them in 250 .mu.L Hank's Balanced Salt Solution, and
cellular number assessed by DNA (DNeasy Tissue Kit, Qiagen
cat#69506).
Quantitation of Osteoblast Differentiation In Vitro.
[0115] For the quantitation of alkaline phosphatase activity, the
inhibitors were prepared in 0.1% DMSO and added to the cultures on
day 1 and every 2-3 days thereafter for 14-16 days. Three different
sets of experiments were performed.
[0116] In the first experimental set, Compound 1 or Compound 2 (0.2
.mu.M or 1 .mu.M) were used as inhibitors with the solvent (DMSO)
as the negative control, and four independent replicate cultures
were prepared at each dose level. The plates were cultured for 14
days. The DNA-normalized activity of alkaline phosphatase is
plotted in FIG. 5.
[0117] The second experimental set was comprised of eight replicate
cultures with Compound 3 (3, 10, and 30M) or Compound 2 (0.1, 0.3,
1.0 .mu.M) as the 15-LO inhibitors. Inhibitors were added either on
Day 1 or Day 11. DMSO was used as the negative control, and
1,25-Vitamin D3 as the positive control for osteoblast
differentiation induction. The normalized activity of alkaline
phosphatase and calcium content is plotted in FIGS. 6 and 7,
respectively. Compound 2 was most effective when added on Day 1,
and Compound 3 was most effective when added on Day 11.
[0118] As a control for the specificity of 15-LO inhibition for the
induction of osteoblast differentiation of stem cells in vitro, the
third set of experiments studied eight replicates of standard
lipoxygenase inhibitors recognized as more specific to the 5-LO
enzyme, rather than the 15-LO enzyme. Zileuton (1, 10, 50 uM,
prepared at Roche as) Compound 5, AA-861 (1, 10, 50 uM, Sigma #
A3711) and Rev-5901 (15 uM, Sigma #R5523) were prepared fresh every
2-3 days and tested in the human osteoblast precursor cells. The
5-LO inhibitors were found to lack ability to induce alkaline
phosphatase activity on day 9 or culture calcium deposition after
16 days of culture, as seen in FIGS. 8 and 9, respectively.
[0119] The results from the quantitation of markers of osteoblast
differentiation, both alkaline phosphatase activity and culture
calcium content, in hMSC cultures stimulated to differentiate into
osteoblasts demonstrate that addition of specific 15-lioxygenase
inhibitors promotes differentiation of the human bone forming cells
in vitro.
[0120] In addition, the ability of 15-lipoxygenase inhibitors to
stimulate bone formation and bone accretion is measured by the use
of clonogenic assays where the clonogenic potential of the bone
marrow precursor cells is measured. The osteoblast and osteoclast
cells are treated with 15-lipoxygenase inhibitors. In one method,
the cells and the inhibitors are incubated in vitro. In the another
method, the mammal is administered the inhibitor, the bone marrow
precursors are isolated, and then plated. For both methods, the
colony forming units derived from these marrow cells is measured.
One can screen for compounds with the potential for increasing bone
mineral density if they are can be shown to increase the osteoblast
clonogeneic potential of the marrow or decrease the osteoclast
clonogeneic potential of the marrow.
Example 4
Disruption of the 15-Lipoxygenase Gene Results in Increased Femoral
Bone Mineral Density
[0121] Initially, 15-LO deficient mice were shown to have minor
phenotypic changes, but subsequently it was shown that these mice
are partially protected from atherosclerotic lesions in a mouse
model (Sun and Funk, J. Biol. Chem., 271, 24055-24062, (1996);
Cyrus et al., J. Clin. Invest., 103, 1597-1604, (1999)). Moreover,
BMD was not measured in 15-LO deficient mice. To examine the effect
of genetic disruption of 15-LO on bone mineral density, 15-LO
"knock-out" (15-LO-KO) mice were compared to parental mice with
respect to bone mineral density. Bone mineral measurements were
determined by dual energy X-ray absorptiometry (DEXA). All studies
were performed with a Lunar PIXImus densitometer (Lunar Corp.,
Madison, Wis.). Whole body densitometric analyses were performed on
anesthetized mice that were 4 months of age when the acquisition of
adult bone mass is complete. The global window was defined as the
whole body image minus the calvarium, mandible, and teeth. After
sacrifice, the right femora were carefully removed and cleaned of
adhering tissue prior to DEXA scanning. Whole body bone mineral
density was not significantly changed between 15-LO-KO and C57BL/6
parental mice (49.8+/-0.6 mg/cm.sup.2 vs. 49.5+/-0.4 mg/cm.sup.2).
Importantly, however, femoral BMD was significantly increased in
the 15-LO knock out mice compared to the C57BL/6 parental strain
(54.0+/-1.2 mg/cm.sup.2 vs. 49.7+/-0.7 mg/cm.sup.2). These results
demonstrate that disruption of 15-LO leads to increased femoral
bone mineral density.
Example 5
15-Lipoxygenase Inhibitors Promote Bone Formation In Vivo
[0122] The ability of 15-lipoxygenase inhibitors to promote bone
formation in vivo was assessed by measuring the ability of the
inhibitors to improve bone parameters in a mouse model of
osteoporosis. Constitutive expression of an Interleukin-4 transgene
from the Lck gene promoter (Lck-IL4) in C57BL/6 mice results in a
severe bone phenotype mimicking osteoporosis. (Lewis et al., Proc.
Natl. Acad. Sci., 90, 11618-22, (1993)) and these mice were used to
demonstrate the effect of treatment with 15-LO inhibitors on bone
mass.
[0123] The four experimental groups used are shown in Table 2. At
the time of weaning, wild type C57BL/6 or transgenic Lck-IL4 mice
received 150 mg/kg bid daily of Compound 2 PD146176 in their food
for 84 days (Diet 5001: 23% protein, 10% fat, 0.95% calcium, and
0.67% phosphorus; PMI Feeds, Inc., St. Louis, Mo.). Mice were
permitted access to one-half the daily food intake twice daily at
approximately 12 h intervals and diet intake was monitored daily.
Water was available ad libitum. The control groups received the
same diet without Compound 2. All groups consumed food equally well
over the course of the experiment. TABLE-US-00003 TABLE 2 Table 2.
Experimental groups used for in vivo inhibitor studies. After
weaning (approximately 28 days), mice were fed chow with or without
15- LO inhibitor (Compound 2) for 84 days. F, female and M, male
animals were equally represented among the groups. C57BL/6 Lck-IL4
transgenic Control Compound 2 Control Compound 2 n = 17 n = 18 n =
20 n = 22 (11F/11M) (8F/9M) (9F/9M) (11F/9M)
[0124] The effects of IL4 overexpression and 15-LO inhibition on
whole body parameters, femoral BMD and geometry, and femoral
biomechanics are shown in Table 3. Over-expression of IL4 results
in a significant decrease in whole body BMD, body weight, and
hematocrit and an increase in percent body fat. Overexpression of
IL4 also reduced femoral BMD, cortical area, moment of inertia, and
cortical thickness compared to wild type C57BL/6 mice. The increase
in marrow area in Lck-IL4 mice correlates with the decreased
hematocrit and associated anemia that has been reported.
Additionally, IL4 overexpression had a significant effect on
femoral biomechanics, including decreased failure load, bone
stiffness and bone strength (Table 3). The mice that were fed the
15-LO inhibitor (Compound 2) showed partial reversal of the
deleterious effects on whole body and femoral bone (Table 3). The
drug-treated mice had a significant increase in whole body BMD,
hematocrit, femoral BMD, and cortical thickness relative to control
mice carrying the Lck-IL4 transgene. Importantly, the 15-LO
inhibitor treated mice had a significant increase in femoral
biomechanics, including failure load, stiffness and strength. This
is particularly relevant to osteoporosis in that the level of bone
strength and failure load are significant determinants in patients
that go on to develop fractures. In summary, the results indicate
that inhibition of 15-LO in vivo leads to a higher BMD and higher
bone strength in an animal model of osteoporosis. TABLE-US-00004
TABLE 3 Table 3. Effects of IL4 overexpression and 15-LO inhibitor
on whole body and femoral bone parameters. Whole body and femoral
bone parameters were measured in mice carrying the Lck- IL4
transgene that leads to overexpression of IL4 compared to C57BL/6
controls. These parameters were also measured in Lck-IL4 mice that
were fed the 15-LO inhibitor Compound 2 compared to untreated
Lck-IL4 controls. NC, Not Changed between groups. ns, p-value was
not significant. Effect of IL-4 Effect of IL-4 Phenotype
overexpression p value overexpression + Compound 2 p value Whole
Body Body Weight -7% 0.042 NC ns % Body Fat +19% 0.007 NC ns
Hematocrit -25% 2 .times. 10-9 +20% 2 .times. 10-5 Whole Body BMD
-11% 2 .times. 10-9 +5% 7 .times. 10-5 Femoral BMD & Geometry
BMD -17% 4 .times. 10-12 +9% 3 .times. 10-6 Length NC ns NC ns
Total area NC ns -4% 0.035 Cortical area -25% 6 .times. 10-12 NC ns
Marrow area +16% 4 .times. 10-7 -7% 7 .times. 10-5 Moment of
Inertia -17% 7 .times. 10-5 NC ns Cortical thickness -27% 9 .times.
10-10 +6% 0.037 Femoral Biomechanics Failure load -45% 6 .times.
10-13 +18% 0.003 Stiffness -48% 3 .times. 10-11 +11% 0.09 Strength
-34% 2 .times. 10-12 +22% 2 .times. 10-4
Animals
[0125] All mice used in the in vivo experiments were bred under
identical conditions at the Portland Va. Veterinary Medical Unit
from stock originally obtained from The Jackson Laboratory (Bar
Harbor, Me.). Breeding mice were maintained for no more than three
generations from stock obtained from The Jackson Laboratory. At the
time of weaning the mice were group housed (9-10 animals per cage)
in a 12 hr light/dark cycle (6:00 AM to 6:00 PM) at 21.+-.2.degree.
C. All procedures were approved by the VA Institutional Animal Care
and Use Committee and performed in accordance with National
Institutes of Health guidelines for the care and use of animals in
research.
Bone Densitometry.
[0126] Bone mineral measurements were determined by dual energy
X-ray absorptiometry (DEXA). All studies were performed with a
Lunar PIXImus densitometer (Lunar Corp., Madison, Wis.). Whole body
densitometric analyses were performed on anesthetized mice that
were 4 months of age when the acquisition of adult bone mass is
complete. The global window was defined as the whole body image
minus the calvarium, mandible, and teeth. After sacrifice, the
right femora were carefully removed and cleaned of adhering tissue
prior to DEXA scanning.
Specimen Processing
[0127] Adult mice (16 weeks old) were euthanized by CO.sub.2
inhalation and weighed to the nearest 0.1 gm. Cardiac blood draws
were obtained immediately upon sacrifice and a small aliquot of
whole blood was reserved for hematocrit determination. Lumbar
vertebrae and both femora were immediately harvested, wrapped in
sterile gauze soaked in phosphate-buffered saline, and stored
frozen at less than about -20.degree. C. for subsequent
analyses.
Femoral Shaft Geometry
[0128] The cross-sectional geometric parameters of the
mid-diaphysis of the femora were determined using a portable x-ray
microtomograph (Model 1074, Skyscan, Antwerp, Belgium). Scans were
taken at 40 mm intervals, and a slice located at the midpoint of
the femur was analyzed for total area (FCSA), cortical area (Ct Ar)
and medullary canal area (Ma Ar). In addition, using the pixels
contained in the cortical region of the digital image, the radius
from the centroid of the cross-section to the outer fiber in the
anterior-posterior plane, the areal moment of inertia (Ixx) in the
plane of bending and the average cortical thickness (Ct Th) were
calculated.
Biomechanical Studies
[0129] The femur was tested to failure in three-point bending on a
high resolution materials test apparatus (Instron Model 4442,
Canton, Mass.). Failure load and stiffness were determined using
system software. Bending strength was then calculated using the
cross-sectional areal measurements previously determined.
Data Analysis
[0130] All data were analyzed by a two-way analysis of variance
(ANOVA) using the JMP software package (SAS Institute).
Example 6
Ability of a 15-lipoxygenase Inhibitor to Increase Bone Formation
In Vivo
[0131] In order to determine the ability of inhibitors of
15-lipoxygenase to stimulate bone formation and bone accretion,
[[[5-(5,6-difluoro-1H-indol-2-yl)-2-methoxyphenyl]amino]sulfonyl]-carbami-
c acid-isobutyl ester, Compound 6, was prepared as described in
WO0196298) and tested in the standard estrogen deficiency Rat OVX
osteopenia assay.
[0132] Three month old rats are ovariectomized (Ovx) and
administered 1 mg/kg/day Compound 6 or 0.1 ug/kg/day 1,25-dihydroxy
vitamin D.sub.3 (positive control, abbreviated Vit. D in Table) by
oral gavage once a day starting at 2 weeks post-ovariectomy and
continuing for 7 weeks. The dose was increased to 2 mg/kg/day and
continued until 11 weeks. Control groups, both sham (rats that were
not ovariectomized) and Ovx, received vehicle only. The bone
mineral density of the spine and right hip was determined by using
the High Resolution Software package on a QDR-4500 Bone
Densitometer.sup..quadrature. (Hologic, Walthan, Mass.). The
animals were scanned by placing them in a supine position such that
the right femur was perpendicular to the main body and the tibia
was perpendicular to the femur. The bone mineral density (gm
mineral/cm.sup.2) for the compounds at the hip and spine are given
in the table below. Animals treated with the 15-LO inhibitor showed
increased BMD in the spine at >3 weeks and the proximal femur at
>7 weeks. Results are shown below. TABLE-US-00005 BMD Treatment
Dose Lumbar Spine Proximal duration Surgery Treatment
.quadrature.g/kg/day L2-L4 L5 Femur 3 weeks Sham Vehicle nd nd nd
Ovx Vehicle 0.2224 0.2343 0.2737 Ovx Vit D 0.1 nd nd nd Ovx Cpd 6
1000 0.2347 (0.055) 0.2480 (0.041) 0.2838 (0.103) 7 weeks Sham
Vehicle .2664 0.2841 0.3068 Ovx Vehicle 0.2303 0.2387 0.2829 Ovx
Vit D 0.1 0.2628 (0.023) 0.2742 (0.015) nd 0.1 ug/kg Ovx Cpd 6 1000
0.2474 (0.019) 0.2549 (0.120) 0.2932 (0.033) 11 weeks Sham Vehicle
0.2740 0.2895 0.3177 Ovx Vehicle 0.2353 0.2525 0.2842 Ovx Cpd 6
2000 0.2507 (0.009) 0.2626 (0.059) 0.2986 (0.039) Values in
parentheses are p values vs OVX control at the same timepoint. nd =
no data
Example 7
A Genetic Manipulation to Reduce 15-lipoxygenase Expression in
Laboratory Mice Results in Improved Bone Mass and Strength
[0133] In order to examine the relationship between 15-lipoxygenase
expression and bone development, a genetically heterogeneous
F.sub.2 population was constructed from two progenitor strains,
B6.129S2-Alox15.sup.tm1Fun (Alox15 knockout or 15LOKO) and DBA/2
(D2), which were purchased from The Jackson Laboratory (Bar Harbor,
Me.). The B6.129S2-Alox15.sup.tm1Fun (15LOKO) mice are homozygous
for a targeted mutation in Alox15 and therefore cannot express
arachidonate 15-lipoxygenase. In contrast, D2 mice exhibit high
levels of Alox15. 15LOKO-D2 F.sub.1 mice were bred locally from
Jackson Laboratory parental lines and intercrossed to generate a
total of 292 15LOKO-D2 F.sub.2 mice. At the time of weaning the
mice were group housed (2-5 animals per cage) and maintained with
ad libitum water and laboratory rodent chow (Diet 5001: 23%
protein, 10% fat, 0.95% calcium, and 0.67% phosphorus; PMI Feeds,
Inc., St. Louis, Mo.) in a 12 hr light/dark cycle (6:00 AM to 6:00
PM) at 21.+-.2.degree. C.
[0134] All mice were studied at 4 months of age when the
acquisition of adult bone mass is complete (28). Bone mineral
measurements were performed with a pencil beam Hologic QDR 1500
densitometer (Hologic, Waltham, Mass.) that was calibrated daily
with a hydroxyapatite phantom of the human lumbar spine. Analysis
was performed using the mouse whole body software (version 3.2),
kindly provided by the manufacturer (Hologic, Waltham, Mass.).
Densitometric analysis was performed on anesthetized mice. Food was
withheld the night prior to examination (to eliminate confounding
effects of undigested rodent chow on bone mineral density
assessment), and the mice were anesthetized by isoflurane
inhalation. The animals were weighed to the nearest 0.1 gm and then
underwent bone density scanning. Mice were euthanized by CO.sub.2
inhalation and spleens and femora were removed aseptically and then
immediately frozen for subsequent analyses. All procedures were
approved by the VA Institutional Animal Care and Use Committee and
performed in accordance with National Institutes of Health
guidelines for the care and use of animals in research.
[0135] Femoral structure (mid-shaft cortical bone area &
cortical thickness) was measured with a desktop x-ray
microtomographic scanner (SkyScan Model 1074, Aartselaar, Belgium).
The left femur was tested to failure in three-point bending with a
high-resolution materials test apparatus (Model 4442, Instron
Corp., Canton, Mass.). An extensometer (Model 2630-113, Instron
Corp.) was attached and system software (Series IX for Windows 95,
Instron Corp.) was used to displace the actuator at a strain rate
of 0.5%/sec. until failure occurred. Load and displacement
(measured using the extensometer) data were recorded and failure
load (F) and stiffness (k, calculated from the linear portion of
the load versus displacement curve) were determined using system
software.
[0136] Genomic DNA was isolated from individual mouse spleens using
a salting-out method. Mice were genotyped with a polymerase chain
reaction (PCR) protocol suggested by The Jackson Laboratory
designed to generate different sized products for the wild type
(266 bp) and mutant (172 bp) Alox15 gene. Amplification was
performed on a Perkin-Elmer 9700 thermocycler (Branchburg, N.J.).
PCR products were separated on 4% agarose gels and visualized with
ethidium bromide staining.
[0137] The 15LOKO-D2 F.sub.2 population was composed of
approximately equal numbers of male (n=141) and female (n=151) mice
and, as is shown in Table 1 below, the Alox15 genotype frequency
conformed to Hardy-Weinberg Principle expectations with 70 (24%)
homozygous knockout (no 15-LO allele) mice, 153 (52%) heterozygous
(single 15-LO allele from D2) mice, and 69 (24%) homozygous D2
(both 15-LO alleles from D2) mice. Although, there was no
difference in body weight, F.sub.2 mice homozygous for the Alox15
knockout exhibited significantly higher whole body BMD than that of
the D2 homozygous or heterozygous mice (p=0.006 by ANOVA).
Furthermore, F.sub.2 mice homozygous for the Alox15 knockout
exhibited increased amounts of femoral shaft cortical bone
(cortical area and cortical thickness) and significantly improved
structural competence as evidenced by increased failure load and
stiffness measures (Table 2). Results are also shown in FIGS.
10-12. TABLE-US-00006 TABLE 1 15LO Genotype Correlates with BMD in
15LOKO-D2 F.sub.2 Population Number of 15LO D2 Alleles ANOVA 0 1 2
p value No. of Mice 69 127 70 Body Weight, g 28.2 .+-. 0.61 27.5
.+-. 0.43 29.2 .+-. 0.64 NS BMD, g/cm.sup.2 66.1 .+-. 0.33 64.9
.+-. 0.25 65.0 .+-. 0.31 p = 0.006
[0138] TABLE-US-00007 TABLE 2 15LO Genotype Correlates with Femoral
Bone Mass and Strength in 15LOKO-D2 F.sub.2 Population Number of
15LO D2 Alleles 0 1 2 p value No. of 15 15 15 Mice Body 23.3 .+-.
0.54 23.9 .+-. 0.45 23.7 .+-. 0.40 NS Weight, g BMD, 65.6 .+-. 0.46
64.8 .+-. 0.95 63.4 .+-. 0.65 p = 0.005 g/cm.sup.2 Ct Ar, 0.76 .+-.
0.03 0.78 .+-. 0.03 0.70 .+-. 0.02 p = 0.046 mm.sup.2 Ct Th, 0.193
.+-. 0.005 0.200 .+-. 0.006 0.176 .+-. 0.004 p = 0.006 mm Failure
20.5 .+-. 0.8 20.3 .+-. 0.8 18.0 .+-. 0.6 p = 0.013 Load, N
Stiffness, 126.5 .+-. 4.6 108.7 .+-. 5.1 110.3 .+-. 3.3 p = 0.004
N/mm (BMD = Whole body BMD; Ct Ar = femoral shaft cortical area; Ct
Th = femoral shaft cortical thickness)
[0139] Thus, novel methods for treating or preventing bone loss,
increasing bone mineral density, and enhancing bone formation and
accretion are disclosed. Although preferred embodiments of the
subject invention have been described in some detail, it is
understood that obvious variations can be made without departing
from the spirit and the scope of the invention as defined by the
appended claims.
* * * * *